Europ. J. CancerVol. 4, pp 305-312. Pergamon Press 1968.
Printed in Great Britain
The Lymph Node Barrier against
Vx2 Cancer Cells before, during and
after Lymphography
A Preliminary Report o f Experiments on Rabbits
U. ENGZELL, C. RUBIO, B. TJERNBERG and J. ZAJICEK
Department of Otolaryngology and Department of Gynaecology, Radiumhemmet, and Department
of Clinical Cytology, Institute of Radiopathology, Karolinska Sjukhuset, Stockholm 60, Sweden
LYMPHOGRAPHY is a well-established diagnostic
procedure. It is used in searching for lymph-
borne metastases of malignant tumours. The
reported complications of the procedure include
wound infection, lymphangitis and oil embolism
to the brain and the lungs [1-4].
The possibility that tumour cells may be
released and transported by the oily contrast
medium during lymphography was studied
by Tjernberg et al. [5] in h u m a n subjects. After
cannulation of the thoracic duct, lymphography
was performed via pedal lymphatics and lymph
was collected from the cannula for cytologic
analysis. In a series comprising 4 cases, no
evidence was obtained that tumour cells were
transported with the contrast medium [6].
The effect of lympography on the barrier
function of lymph nodes has received little
attention in the literature. It is conceivable that
the lymph node barrier against cancer cells is
disturbed by lymphography, thus permitting
lymphatic dissemination of the tumour during
or after lymphography. An investigation of the
possible effect of lymphography on the barrier
function of lymph nodes seemed therefore to be
warranted.
The most appropriate procedure in such an
investigation involves cannulation of afferent
and efferent lymphatics of a single node,
injection of contrast medium and cancer cells
into the afferent vessel and collection of
305
efferent fluid for cytologic analysis. Experiments
of this type obviously cannot be made on human
subjects.
In the present study rabbits were used.
Vx2 carcinoma cells were injected into a
lymphatic entering the popliteal lymph node
before and after lymphography. The barrier
function of the node was assessed by studying
the efferent lymph for presence of the Vx2 cells.
A description of the technique is now presen-
ted along with a preliminary report of findings
in 16 rabbits.
MATERIALS AND METHODS
Cannulation of lymphatics
Simultaneous cannulation of afferent and
efferent vessels of single lymph nodes has previ-
ously heen reported by only a few workers.
Drinker et al. [7] and Widdicombe et al. [8]
used the method for studying the barrier
function of isolated lymph nodes in dogs and
rabbits, respectively, against bacteria and
erythrocytes. Sabiston et al. [9] cannulated
afferent and efferent lymph vessels in investi-
gations of the lymphatico-venous pathways in
isolated lymph nodes in the neck of dogs.
Hall and Morris [10] used a similar technique
in sheep to observe changes in the cell popu-
lation of efferent lymph after injection of
antigen into afferent vessels. Fisher and
Fisher [11] applied the procedure in studies
306 U. Engzell, C. Rubio, B. Tjernberg and J. Zajicek
of the barrier function of normal popliteal
lymph nodes in rabbits against transplantable
tumour cells.
O u r investigation of the barrier function of
popliteal lymph nodes against cancer cells in
connection with lymphography was performed
on full-grown domestic rabbits of both sexes.
The rabbits were anaesthetized via a polythene
catheter (PE 50, Clay-Adams Co. Inc.) inserted
into a marginal ear vein. Veterinary Nembutal
(Abbott 60 mg/ml) diluted 1:5 in 0 . 9 %
NaC1 was used. By leaving the catheter in
position throughout the experiment, mainten-
ance doses could be given as required.
The lateral aspect of the thigh was shaved
and cleaned and the popliteal fossa was explored
through an incision into skin and muscle. The
rabbit has a single lymph node in the popliteal
fossa. This node has four to six afferent lymph
vessels and as a rule a single efferent vessel,
which is easily identifiable under a dissection
microscope. If two efferent vessels were seen
the experiment was discontinued.
The efferent lymph vessel was carefully freed
from its surrounding tissues. A polythene
catheter (PE 50), having at its tip a small cuff,
was inserted into this vessel and was fixed with
a ligature. The cuff was formed by heating the
catheter tip over a flame [12]. The afferent
lymphatics were visualized by injection of
about lml of Patent Blue Violet (Gurr & Co.)
into the paw. The most prominent of the
afferent vessels was then threaded with a metal
cannula or a polythene catheter (PE 10)
introduced in proximal direction close to the
node and fixed with a ligature (Fig. 1). To
permit insertion of the catheter, its tip had to
be thinned out by pulling it over a flame as
recommended by Fisch [13].
The cannulations were done under a Zeiss
dissection microscope and at 6 - 1 6 m a g n i -
fication. In order to test the system for leakage,
1 ml of 0 . 9 % NaC1 was injected into the affer-
ent lymphatic and the fluid from the efferent
vessel was collected in a test tube.
Cannulation of afferent and efferent lym-
phatics was successfully performed in 16 experi-
ments. After a little practice, we found no
difficulty in cannulating efferent lymphatics,
but the greater difficuties presented by the
finer afferent vessels constituted the main
reason for lack of success in 6 experiments.
Preparation of cell suspensionsfor infusion
A prerequisite for studying the barrier
function of lymph nodes is that the cells which
may penetrate the barrier should be readily
identifiable. Tjernberg and Zajicek [14] found
unmistakable malignant cells in efferent lymph
after digital massage of lymph nodes with meta-
static Vx2 carcinoma. A search of the literature,
however, has revealed no investigation on
whether or not individual cells of Vx2 carci-
noma can always be confidently distinguished
on morphologic grounds (cell size, cytoplasmic:
nuclear ratio, etc.) from the cells normally
present in the lymph.
We therefore performed morphologic studies
on Vx2 carcinoma cells of two types. These
cells were obtained either directly by aspiration
biopsy of transplantable tumours growing in
domestic rabbits (transplantable tumour cells,
Fig. 2) or were taken from tissue cultures
(cultured tumour cells, Fig. 3).
Tissue cultures of Vx2 carcinoma cells were
prepared by one of us (C. Rubio) as follows.
A biopsy specimen measuring approximately
2 cma was taken from a 1-month-old transplan-
ted Vx2 tumour in a rabbit and was immersed
in sterile Early medium complemented with
L-glutamine, antibiotics and 20% bovine
serum. Using sterile technique, the specimen
was transferred to a Petri dish and areas of
suspected necrosis were removed. The specimen
was now washed in various changes of the
mentioned medium and was chopped until a
creamy material was obtained. After addition
of 100 ml trypsin ( 1 % in trypsin buffer SBL),
the specimen was incubated under constant
magnetic stirring for ten minutes. The super-
natant was discarded. The sediment was
resuspended in 100 ml trypsin solution and the
suspension was incubated for an additional
40 rain. In order to remove clumps, the
suspension was sieved through four layers of
surgical gauze.
The percolated material was centrifuged at
room temperature (1000 rev/min for 3 minutes)
and the resulting sediment was resuspended in
fresh medium. From this suspension 12 106
trypan-blue negative cells were transferred in
15 ml of the medium to stoppered milk flasks.
After a lag period of three weeks, the cultures
entered into a phase of growth. Sixteen passages
were then made before the cells were ready for
use in our experiments.
These cultured Vx2 carcinoma cells were
suspended in rabbit lymph which had been
rendered acellular by centrifugation. The
transplantable Vx2 cells obtained directly by
aspiration biopsy were suspended in the same
way.
The tumour cells were sedimented by centri-
fugation of the suspension. They were then
washed in 0.9 % NaC1 and fixed with methanol-
acetic acid mixture [14]. To a drop of fixed
Fig. 1. Lymphogram showing the popliteal node in a rabbit. A polythene catheter has been inserted into
the efferent vessel and a metal cannula into an afferent lymphatic.

Fig. 2. Slide prepared from Vx2 carcinoma cells aspirated from a tumour growing zn the hind paw of a
rabbit. Harris' haematoxylin stain x 480.

(to face p. 306)

 0
0 o 0
o

0 0 6
%
F
0
0

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Fig. 3. Cultured Vx2 carcinoma cells spread on a slide after trypsinization and washing. Harris'
haematoxylin stain 480.

at 0
D Q

. ,% ,.
a
OgQ e O"Q O ~
0 0 O
,g

O0

~ ~:i!~iI ~iii~ ~i~~ ~ ~ O

~/51~iOiiii~~~i~~,~ ~ii!iii~!i~ !i;ii~~0!i ~ i~i~i~.... ~ ~ ~
~ ~~ii :~ :~ i ~ i!~~ i~ ~ii ~ ~
~i~~I~i~i ~i,!i"~!i'i!i!!ii i!:i!:~!~
~~ ~!!: ~!!~

Fig. 6. Cultured Vx2 carcinoma cells collectedfrom the efferent popliteal lymphatic. The cells had been
injected into the afferent lymphatic o[ the same node after lymphography with Lipiodol Ultrafluid. Harris'
haematoxylin stain 480.
Fig. 7. Section from a popliteal lymph node after lymphography with oily contrast medium (Lipiodol
Ultrafluid). Sinuses are greatly distended by the medium. Haematoxylin-eosin stain 75,6.

Fig. 8. Section from a popliteal lymph node after lymphography with water-soluble contrast medium
(Urografin) and subsequent infusion of cultured Vx2 carcinoma cells into the afferent lymphatic. The
carcinoma cells have accumulated in subcapsular sinuses. Haematoxylin-eosin x 270.
 Vx2 Cancer Cells 307

25-

20-
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10-
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00000

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5-
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10 20
Number of rabbits

Fig. 4. Percentagesof large lymphoid cells (dia. ~ 8 la) in efferent lymph from the popliteal node in 20
rabbits (filled circles) and in fluid leaving the node after perfusion with 1 ml 0 . 9 % NaC1 in 13 rabbits
(open circles).
The rabbits are arranged in order offrequency of large cells in the efferent lymph.

50-

.................... Large lymphoid cells

. . . . Transplantable Vx2 carci-
n o m a cells
40- Cultured Vx2 carcinoma
cells

=) 3 0 -
u

E
:~ 2 0 -

lO-

//
I I [
10 2O 3O
Cell diameter in microns

Fig. 5. Size distribution curvesfor large lymphoid cells (diameter~. 8 It), aspirated Vx2 carcinoma cells
and cultured Vx2 carcinoma cells.
308 U. Engzell, C. Rubio, B. Tjernberg and J. Zajicek
carcinoma cells on a glass slide a drop of 1%
pectin adhesive solution [15] was added and
mixed well. The slides were dried in air and
then stained with haematoxylin-eosin. Slides
were prepared in the same way from cells
sedimented from efferent lymph.
Comparisons of the slides showed that small
lymphoid cells (diameter up to 8g) could
readily be distinguished from both aspirated
and cultured Vx2 carcinoma cells by virtue of
morphologic details in the cytoplasm or nuclei.
Possible exceptions were lymphocytes with
pyknotic nuclei, which were occasionally found
in the efferent lymph and closely resembled
pyknotic, small Vx2 carcinoma cells in aspirate.
When cell diameter was 8g or more, however,
differentiation on the same morphologic basis
between lymphoid cells and aspirated Vx2
carcinoma cells was difficult. The frequency
of such large lymphoid cells among 200 con-
secutive cells in the lymph leaving a popliteal
node was determined in 20 healthy rabbits.
In 13 of them the fluid leaving the node after
perfusion with 1 ml 0 . 9 % NaC1 was also
analyzed. The results are shown in Fig. 4.
Lymphoid cells whose diameter was at least 8g
formed up to 22% of the total cell population
in the undiluted lymph (filled circles) and up
to 9% in the efferent fluid after NaC1 perfusion
(open circles).
Since individual cells of transplantable Vx2
carcinoma were not reliably distinguishable
from the larger cells commonly occurring in
normal efferent lymph, the possibilities offered
by the cultured Vx2 cells were next explored.
These cells appeared generally to be larger
than the Vx2 cells aspirated from transplant-
able tumours, and therefore the feasibility of
basing the differentiation on cell size was
investigated.
For this purpose, micrometric determinations
were made on consecutively observed cells--
100 Vx2 cells from aspirates and 100 from
cultures and also 100 lymphoid cells with a
diameter of at least 8p. Figure 5 presents the
size distribution curves with 95% tolerance
limits at 95% confidence levels. The limits for
the curve representing large lymphoid cells are
wholly within those of the curve for aspirated
Vx2 carcinoma cells. The tolerance limits were
calculated from the formula 2 ~ 2 " 233 s, where
is the mean and s the standard deviation.
Lower limit Upper limit
Large lymphoid cells 6-99 11.07
Aspirated Vx2 carcinoma cells 4.45 17- 39
Tissue-cultured Vx2 cells 7.97 34.40
The numerical calculations of the tolerance
limits were as follows.
From these studies it was concluded that
individual Vx2 cells aspirated from trans-
plantable tumours cannot always be distin-
guished from large lymphoid cells on mor-
phologic grounds, including cell size. Cultured
Vx2 carcinoma cells, however, are larger than
the cells in efferent lymph and thus are more
easily identified. Consequently, cultured Vx2
carcinoma ceils were used in the present
investigation on the barrier function of lymph
nodes in connection with lymphography.
Intralymphatic infusion of cultured Vx2 carcinoma
cells
The cells were taken from culture flasks and
after trypsinization were washed twice in
Hanks' balanced salt solution. Concentrations
of 1.5-2 106 cancer ceils in 1 ml of Hanks'
solution were used for infusion. One ml of the
cell suspension was injected manually, using
an ordinary plastic syringe, into a cannulated
afferent vessel over a period of 20 to 30 minutes.
In this way 5 0 , 0 0 0 - 100,000 cancer cells per
min. entered the lymph node. During the
infusion the syringe was repeatedly shaken in
order to minimize sedimentation of the cells.
The efferent lymph was collected in sili-
conized tubes which contained 2 drops of
heparin. The tubes were immersed in an
icebath ( + 4 C ) .
Infusion of O. 9 % NaCl
After infusion of the Vx2 cancer cell suspen-
sion, 1 ml of 0 . 9 % NaC1 solution was injected
into the same vessel and the efferent fluid was
collected as before for cytologic analysis.
Lymphography
After infusion of 1 ml of 0.9//o NaC1 the
contrast medium was injected into the afferent
lymphatic over a period of 15 to 25 minutes.
When a few drops of the contrast medium
were recovered from the efferent vessel, the
injection was discontinued. The volume of
injected contrast medium was about 0 . 2 -
0.3 ml. Lipiodol Ultrafluid (Laboratories
Andrd Guerbet, France) was used in 6 cases,
Urografin 60% (Schering A. G., Berlin) in 3
cases and Emulsion Leo 372 (Leo A. B., Sweden)
in 5 cases.
Post-lymphography infusion of 0 . 9 % NaCl and
of cultured Vx2 carcinoma cells
When contrast medium appeared in the
efferent lymph, 1 ml of an 0 - 9 % NaC1 solution
was injected over 10 to 15 minutes into the
afferent vessel in 12 cases. Efferent fluid was
collected for cytologic analysis as previously

Table 1. Presence of cultured Vx2 carcinoma cells in efferent lymph before, during and after
lymphography. Experimental study on the popliteal node in 16 rabbits
Injection into an afferent No. of
lymphatic rabbits
Cancer cells in Hanks' solution 16
0.9% NaC1 solution 14
Contrast medium 14
0" 9% NaC1 solution 12
Cancer cells in Hanks' solution 11
described. Inspection o f this fluid indicated
t h a t some o f the contrast m e d i u m was r e m o v e d
f r o m the l y m p h n o d e d u r i n g the NaC1 injection.
As a final step in the e x p e r i m e n t a l p r o c e d u r e ,
a suspension o f 1 - 5 - 2 106 Vx2 c a n c e r cells
in 1 ml o f H a n k s ' solution was injected in 11
cases a n d efferent l y m p h was again collected
for cytologic analysis.
Cytologic processing
F r o m the samples collected from efferent
l y m p h vessels the cells were isolated as follows.
W h e n Lipiodol h a d b e e n injected, 1 ml o f
0 . 9 % NaC1 solution was a d d e d to the sample.
Because o f its higher specific gravity as com-
p a r e d with l y m p h or lymph-NaC1, the L i p i o d o l
settled to the b o t t o m o f the tube. T h e super-
n a t a n t was p i p e t t e d off a n d the cells were
isolated b y centrifugation.
W h e n Emulsion Leo, U r o g r a f i n or N a C I
solution h a d b e e n injected, the sample was
first centrifuged at 1500 r e v / m i n for 10 minutes.
T h e s u p e r n a t a n t was p i p e t t e d off a n d the
sediment was washed twice in NaC1 solution
b y centrifugation.
T h e sedimented cells were i m m e d i a t e l y fixed
with m e t h a n o l - a c e t i c acid a n d allowed to
stand overnight. T h e fixed cells were centri-
fuged at 1500 r e v / m i n for 7 minutes a n d the
sediment was transferred to p r e c l e a n e d slides.
T h e b o t t o m of the e m p t i e d centrifuge t u b e was
rinsed with a d r o p o f 1% pectin solution,
w h i c h was t h e n a d d e d to the slides to increase
adhesion o f the cells to the glass. T h e slides
were stained with Harris h a e m a t o x y l i n for
one m i n u t e a n d m o u n t e d with Eukitt synthetic
resin.
Vx2 Cancer Cells 309
Cytologic findings in efferent lymph
No cancer cells Cancer cells
(no. of rabbits) (no. of rabbits)
15
14
13 1 of 6 injected with Lipiodol
0 ,, 5 ,, ,, Emulsion Leo 372
0 ,, 3 . . . . Urografin 60%
10 1 ,, 4 ,, ,, Lipiodol
0 ,, 5 ,, ,, Emulsion Leo 372
1 ,, 3 .... Urografin 60%
3 " 3 .... Lipiodol
2 ,, 5 ,, ,, Emulsion Leo 372
1 ,, 3 ,, ,, Urografin 60%
RESULTS
T a b l e 1 summarizes the experiments a n d
the results o f the cytologic analyses.
It is seen t h a t following injection o f c u l t u r e d
Vx2 c a r c i n o m a cells into an afferent l y m p h a t i c
o f the popliteal node, c a n c e r cells were detected
in the efferent l y m p h in only 1 o f the 16 rabbits.
W h e n NaC1 solution was thereafter injected
into the afferent l y m p h a t i c in 14 of these 16
rabbits, none o f t h e m was f o u n d to h a v e c a n c e r
cells in the efferent l y m p h f r o m this node. T h e
reason for w i t h d r a w a l of 2 rabbits at this stage
o f the experiments was difficulty in keeping
the c a n n u l a system running. O n e o f the 2
rabbits was t h a t m e n t i o n e d as h a v i n g c a n c e r
cells in efferent l y m p h .
I n the r e m a i n i n g 14 rabbits the experiments
were c o n t i n u e d b y injecting contrast m e d i u m
into the afferent l y m p h vessel. O n l y one o f the
samples collected after passage of contrast
m e d i u m t h r o u g h the l y m p h n o d e c o n t a i n e d
c a n c e r cells ( T a b l e 1). T h e m e d i u m in this case
was Lipiodol Ultrafluid. I n the same rabbit,
severe bleeding could be seen in the l y m p h n o d e
a n d the efferent l y m p h also was h a e m o r r h a g i c .
Perfusion of the l y m p h n o d e with contrast
m e d i u m was followed b y injection o f 1 ml NaC1
in 12 o f the 14 rabbits. ( T h e 2 other rabbits
were w i t h d r a w n because of leakage in the
system. Both h a d been injected with Lipiodol).
T h e fluid collected from the efferent l y m p h a t i c
after this NaC1 injection was f o u n d to contain
contrast m e d i u m . T h e fluid c o n t a i n e d c a n c e r
cells in the r a b b i t in which such cells h a d
already b e e n detected after passage o f Lipiodol,
a n d also in 1 o f the 3 rabbits which h a d been
injected with Urografin. N o Vx2 c a r c i n o m a
310 U. Engzell, C. Rubio, B. Tjernberg and J. Zajicek
cells were found in the efferent lymphatic
fluid collected from the other 10 rabbits
(Table 1).
A suspension of cancer cells was finally
injected into the afferent lymph vessel in 11
of the 12 rabbits. (One rabbit was withdrawn
because of leakage in the system.) In 5 cases,
no malignant cells appeared in the efferent
fluid. Numerous cancer cells were found in this
fluid in all 3 rabbits which had been injected
with Lipiodol (Fig. 6). A few cancer cells were
detected in 2 of the 5 rabbits in which Emulsion
Leo had been used and in 1 of the 3 injected
with Urografin.
DISCUSSION
The filtering action of lymph nodes on
tumour cells was recognized more than a
century ago by Virchow [16], who discussed
this action in connection with lymph-borne
metastases of malignant m a m m a r y tumours.
The lymph node barrier against malignant
cells has previously been studied along two lines.
Some writers noted the occurrence of distant
metastases after intralymphatic tumour-cell
injection [17-19]. Others attempted a more
direct approach by recording the presence of
tumour cells in efferent lymphatic vessels
[I 1, 20].
Pressman et al. [20] performed intranodal
injection of a highly concentrated suspension
of H e L a cells and then found such cells in the
efferent lymph. But this technique may be
traumatic and therefore may create abnormal
communications in the injected node [9].
Intralymphatic and subcutaneous injection of
Vx2, Brown-Pearce and Walker carcinoma
cells into the paw in rabbits was reported by
Fisher and Fisher [11] to be followed within
60 minutes of the injection by appearance of
tumour cells in the efferent lymph from popliteal
nodes. When we tried to reproduce their
experiments in 6 rabbits by subcutaneous injec-
tion of Vx2 carcinoma cells into the paw, no
significant change occurred in the cell popu-
lation of the efferent lymph as compared with
control rabbits.
One of the prerequisities for investigating
the lymph node barrier function by recording
the presence of tumour cells in efferent lymph
is the possibility of differentiating tumour cells
from the cells that are ordinarily present in
the lymph. We found difficulty in making
reliable distinction between individual Vx2
carcinoma cells obtained by aspiration biopsy
and large lymphoid cells. But cultured Vx2
carcinoma cells were more easily distinguishable
from normal constituents of the lymph, par-
ticularly because of their larger size, and
therefore were better suited for such experimen-
tal work.
The results of these experiments showed that
the lymph node barrier, as a rule, is effective
against cultured Vx2 carcinoma cells, free or
forming clusters, injected into an afferent
lymphatic. The carcinoma cells thus were
detected in efferent lymph in only 1 of the 16
tested rabbits. Whether in this rabbit the barrier
was damaged during the injection or whether
the cells escaped through some pre-existing
direct communication between the afferent
and efferent lymphatics is not known, however.
The main purpose of our study was to evolve
an experimental procedure by which the
possibility of tumour-cell dissemination in
lymphography using oily or water-soluble
contrast medium could be assessed. By intro-
ducing freely suspended clustered or individual
carcinoma cells into a lymph node via an
afferent lymphatic, we created presumably
optimal conditions for spreading these cells
during or after subsequent lymphography.
Two aspects of the problem could thus be
studied, viz., the possible transportation of
cancer cells by contrast medium in its passage
through a lymph node, and the barrier function
of that node against cancer cells before and
after lymphography.
The observations hitherto made indicate that
transportation of tumour cells by contrast
medium is not a common phenomenon in
lymphography. When injection of cancer cells
(free or in cluster) into an afferent lymphatic
was followed by injection of contrast medium
into the same vessel, cancer cells were found in
the efferent contrast-containing lymph in only 1
of 14 rabbits. During its passage through the
lymph node the contrast medium apparently
did not attract malignant cells previously
deposited in the node. It is pertinent that in
suspensions which contained, in addition to
lymphocytes or Vx2 carcinoma cells, droplets
of Lipiodol Ultrafluid or Emulsion Leo 372,
there was no discernible mutual adhesion
between the cells and the oily contrast medium.
Does lymphography damage the structure
of lymph nodes in such a way that their barrier
function against passage of tumour cells is
impaired ?
Lymphography with Lipiodol Ultrafluid
seemed to cause complete breakdown of the
lymph node barrier, since subsequently injected
Vx2 cells were found in large numbers in the
efferent lymph in all of the tested rabbits.
Emulsion Leo 372, which contains microscopic
droplets of oily medium, possibly was less
 Vx2 Cancer Cells 311

traumatic: The efferent lymph contained a ments is that it is inadvisable to remove

few carcinoma cells in only two of the five
rabbits studied in the same way. The water-
soluble Urografin likewise seemed to be
relatively nontraumatic [Figs. 7 and 8].
The observations concerning Lipiodol Ultra-
fluid may, however, be inapplicable to clinical
lymphography in man. The contrast medium
then remaining in the lymph nodes is slowly
absorbed over a period of weeks or months.
The only conclusion permitted by our exper-
contrast medium immediately after lympho-
graphy by perfusion of the nodes, for example
with chemotherapeutic agents, if there is any
suspicion that the area which drains towards
the nodes still contains cancer cells. Post-
lymphography perfusion of lymph nodes may
conceivably open up communications blocked
by oily contrast medium and cause dissemina-
tion of the tumour from a primary site through
the perfused nodes.

RESUME
L'effet de la lymphographie sur la fonction des ganglions lymphatiques, comme barri~re
vis-&vis des cellules carcinomateuses, a itl gtudig, par la cathgt#isation des lymphatiques
affgrents et efferents du ganglion poplitg, chez le lapin. Des ceUules carcinomateuses Vx2
cultivles gtaient injectges dans le vaisseau affgrent, avant et aprks lymphographie par le
Lipiodol Ultrafluide, l'Emulsion Leo 372, ou l'Urographine. Ces cellules gtaient
facilement reconnaissables parmi les cellules du ganglion. Les modifications de la fonction
de barri#e du ganglion gtaient estimges par l'analyse cytologique du liquide recueilli au
niveau du vaisseau lymphatique effgrent.

Les rgsultats prdliminaires indiquent :

(1) que le ganglion est une barri#e efficace vis-&vis des cellules carcinomateuses Vx2
cultivges, injectges dam le vaisseau affgrent;
(2) que les cellules Vx2, introduites dans un ganglion, avant la lymphographie, ne sont pas
emportges par le produit de contraste lors de son passage ~ travers le ganglion;
(3) que la fonction de barri~re du ganglion est diminude apr?s lymphographie, particulik-
rement avec le Lipiodol Ultrafluide: des ceUules carcinomateuses Vx2, injectles apr~s
lymphographie, se retrouvaient dam le liquide lymphatique effgrent.

SUMMARY
In order to study the effect of lymphography on the function of lymph nodes as a barrier
to carcinoma cells, cannulation of afferent and efferent lymphatics of the popliteal node
was performed in domestic rabbits. Cultured Vx2 carcinoma cells were injected into an
afferent vessel before and after lymphography with Lipiodol Ultrafluid, Emulsion Leo
372 or Urografin. These cells were readily recognizable among lymph cell populations.
The changes in the barrier function of the node were judged by cytologic analysis of fluid
collectedfrom the cannulated efferent lymphatic.
The preliminary results indicated:
(1) that the lymph node is an effective barrier against cultured Vx2 carcinoma cells
injected into an afferent lymphatic;
(2) that Vx2 carcinoma cells introduced into a node prior to lymphography are not tram-
ported by the contrast medium in its passage through the node, and
(3) that the barrier function of lymph nodes is impaired after lymphography, especially
with Lipiodol Ultrafluid : Vx2 carcinoma cells injected after lymphography were found in
the efferent lymph.

ZUSAMMENFASSUNG
Um die Auswirkung der Lymphographie auf die Funktion der Lymphknoten als Barriere
fiir KarzinomzeUen zu untersuchen, wurden die afferenten und efferenten LymphgefiiJ3e
yon Lymphknoten in der Poplitea bei Kaninchen kaniiliert. Kulturen yon Vx2-Karzinom-
zeUen wurden in die afferenten Gefiifle injiziert und zwar vor und nach Lymphographie
mit Lipiodol Ultrafluid, Emulsion Leo 372 oder Urografin. Diese Zellen waren leicht
erkennbar innerhalb der Lymphknotenzellpopulation. Veriinderungen in der Barrieren-
312 U. Engzell, C. Rubio, B. Tjernberg and J. Zajicek

funktion der Knoten wurden beurteilt durch eine zytologische Analyse der Fliissigkeit, die
aus der kannulierten efferenten Bahn gesammelt wurde.
Die vorliiufigen Ergebnisse zeigen, daft erstens der L~mphknoten eine wirksame Barriere
gegen kultivierte Vx2-Karzinomzellen ist, die in das afferente Lymphgefiifl injiziert
werden. Zweitens, daft Vx2-Karzinomzellen, die in einen Knoten vor der Lymphographie
injiziert werden, nicht durch das Kontrastmedium bei seiner Passage durch den Lymph-
knoten mitgeschleppt werden, und drittens, daft die Barrierefunktion von Lymphknoten
nach der Lymphographie verschlechtert ist, speziell bei Verwendung von Lipiodol Ultrafluid.
Vx2-Karzinomzellen, die nach der Lymphographie injiziert wurden, konnten in den
efferenten LymphgefiiJ3en gefunden wereden.

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